Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

National Institutes of Health

The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However, the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study, we used quantitative real-time PCR, Northern blots, whole genome RNA sequencing, Western blots, and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full-length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However, ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover, surface ABCG2 protein was coexpressed with the differentiation marker stage-specific embryonic antigen-1 of hESCs, following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the downregulation of two microRNAs (miRNAs) (i.e., hsa-miR-519c and hsa-miR-520h). Transfection of miRNA mimics and inhibitors of these two miRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by miRNAs in hESCs.

Regulation and expression of the ATP-binding cassette transporter ABCG2 in human embryonic stem cells.

Regenerative medicine, relying on human embryonic stem cell (hESC) technology, opens promising new avenues for therapy of many severe diseases. However, this approach is restricted by limited production of the desired cells due to the refractory properties of hESC growth in vitro. It is further hindered by insufficient control of cellular stress, growth rates, and heterogeneous cellular states under current culture conditions. In this study, we report a novel cell culture method based on a non-colony type monolayer (NCM) growth. Human ESCs under NCM remain pluripotent as determined by teratoma assays and sustain the potential to differentiate into three germ layers. This NCM culture has been shown to homogenize cellular states, precisely control growth rates, significantly increase cell production, and enhance hESC recovery from cryopreservation without compromising chromosomal integrity. This culture system is simple, robust, scalable, and suitable for high-throughput screening and drug discovery.

Non-colony type monolayer culture of human embryonic stem cells.

&#x5F88;&#x591A;&#x7531;&#x8BF1;&#x5BFC;&#x7684;&#x591A;&#x529F;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x6280;&#x672F;&#x751F;&#x6210;&#x7684;&#x5174;&#x594B;&#x4E0E;&#x5EFA;&#x6A21;&#x4EE5;&#x53CA;&#x6F5C;&#x5728;&#x7684;&#x4E2A;&#x6027;&#x5316;&#x7684;&#x7EC6;&#x80DE;&#x6CBB;&#x7597;&#x7684;&#x75BE;&#x75C5;&#x7684;&#x53EF;&#x80FD;&#x6027;&#x6709;&#x5173;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x8981;&#x8FFD;&#x6C42;&#x8FD9;&#x662F;&#x91CD;&#x8981;&#x7684;&#x662F;&#x8981;&#x4E86;&#x89E3;&#x5305;&#x62EC;&#x5176;&#x56FA;&#x6709;&#x53D8;&#x5F02;&#x6027;&#x591A;&#x80FD;&#x5E72;&#x6B63;&#x5E38;&#x72B6;&#x6001;&#x3002;&#x6211;&#x4EEC;&#x6267;&#x884C;&#x5404;&#x79CD;&#x5206;&#x5B50;&#x5206;&#x6790;&#x5316;&#x9A8C; 21 hESC &#x7EBF;&#x6761;&#x548C; 8 hiPSC &#x884C;&#x6765;&#x751F;&#x6210;&#x5168;&#x9762;&#x7684;&#x591A;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x672A;&#x5206;&#x5316;&#x72B6;&#x6001;&#x7684;&#x5FEB;&#x7167;&#x3002;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x6570;&#x636E;&#x7684;&#x5206;&#x6790;&#x663E;&#x793A;&#xFF0C;&#x6839;&#x636E;&#x524D;&#x51E0;&#x6B21;&#x62A5;&#x544A;&#x6CA1;&#x6709;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x7279;&#x5F02;&#x6027;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x6A21;&#x5F0F;&#x3002;&#x6211;&#x4EEC;&#x8FDB;&#x4E00;&#x6B65;&#x6BD4;&#x8F83;&#x5206;&#x5316;&#x4F5C;&#x4E3A;&#x80DA;&#x4F53; 2 &#x5A92;&#x4F53;&#x62DF;&#x542F;&#x52A8;&#x5BF9;&#x5355;&#x72EC;&#x7684;&#x5355;&#x5143;&#x683C;&#x7684;&#x547D;&#x8FD0;&#xFF0C;&#x4EE5;&#x53CA; 20 &#x6210;&#x4EBA;&#x7EC4;&#x7EC7;&#x5206;&#x5316;&#x7684;&#x7EC6;&#x80DE;&#x3002;&#x4ECE;&#x8FD9;&#x4E00;&#x5206;&#x6790;&#x6211;&#x4EEC;&#x5DF2;&#x7ECF;&#x4EA7;&#x751F;&#x4E86;&#x57FA;&#x56E0;&#x5217;&#x8868;&#x5B9A;&#x4E49;&#x591A;&#x6F5C;&#x80FD;&#xFF0C;&#x4E3A;&#x591A;&#x80FD;&#x5E72;&#x56FD;&#x5BB6;&#x5EFA;&#x7ACB;&#x4E00;&#x4E2A;&#x57FA;&#x7EBF;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x63D0;&#x4F9B;&#x5217;&#x8868;&#x663E;&#x793A;&#x72EC;&#x7ACB;&#x5355;&#x5143;&#x683C;&#x547D;&#x8FD0;&#x7684;&#x62DF;&#x8BAE;&#x7684;&#x504F;&#x89C1;&#x8FD9;&#x4E24;&#x79CD;&#x5206;&#x5316;&#x6761;&#x4EF6;&#x4E30;&#x5BCC;&#x7684;&#x57FA;&#x56E0;&#x3002;

StemCellDB&#xFF1A; &#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x6570;&#x636E;&#x5E93;&#x5728;&#x56FD;&#x5BB6;&#x5065;&#x5EB7;&#x7814;&#x7A76;&#x6240;&#x3002;

Much of the excitement generated by induced pluripotent stem cell technology is concerned with the possibility of disease modeling as well as the potential for personalized cell therapy. However, to pursue this it is important to understand the 'normal' pluripotent state including its inherent variability. We have performed various molecular profiling assays for 21 hESC lines and 8 hiPSC lines to generate a comprehensive snapshot of the undifferentiated state of pluripotent stem cells. Analysis of the gene expression data revealed no iPSC-specific gene expression pattern in accordance with previous reports. We further compared cells, differentiated as embryoid bodies in 2 media proposed to initiate differentiation towards separate cell fates, as well as 20 adult tissues. From this analysis we have generated a gene list which defines pluripotency and establishes a baseline for the pluripotent state. Finally, we provide lists of genes enriched under both differentiation conditions which show the proposed bias toward independent cell fates.

StemCellDB: The Human Pluripotent Stem Cell Database at the National Institutes of Health.

Une grande partie de l&#39;enthousiasme g&eacute;n&eacute;r&eacute; par la technologie des cellules souches pluripotentes induites est pr&eacute;occup&eacute;e par la possibilit&eacute; de maladie de mod&eacute;lisation ainsi que le potentiel de la th&eacute;rapie cellulaire personnalis&eacute;. Toutefois, afin de poursuivre ce, il est important de comprendre l&#39;&eacute;tat de pluripotentes &laquo; normal &raquo;, y compris sa variabilit&eacute; inh&eacute;rente. Nous avons effectu&eacute; plusieurs essais mol&eacute;culaires de profilage pour 21 lign&eacute;es de CSEh et 8 lignes de hiPSC pour g&eacute;n&eacute;rer un aper&ccedil;u complet de l&#39;&eacute;tat indiff&eacute;renci&eacute; de cellules souches pluripotentes. Analyse les donn&eacute;es d&#39;expression g&eacute;nique a r&eacute;v&eacute;l&eacute; aucun mod&egrave;le d&#39;expression sp&eacute;cifiques iPSC g&egrave;ne conform&eacute;ment aux pr&eacute;c&eacute;dents rapports. Plus loin, nous avons compar&eacute; les cellules diff&eacute;renci&eacute;es comme corps embryo&iuml;des dans 2 m&eacute;dias propos&eacute;s de lancer la diff&eacute;renciation vers des destins cellulaires distincts, ainsi que 20 tissus adultes. De cette analyse, nous avons g&eacute;n&eacute;r&eacute; une liste de g&egrave;ne qui d&eacute;finit la pluripotence et &eacute;tablit une ligne de base pour l&#39;&eacute;tat pluripotent. Enfin, nous fournissons des listes de g&egrave;nes enrichis dans les deux conditions de diff&eacute;renciation qui montrent le biais propos&eacute; vers les destins de cellules ind&eacute;pendantes.

StemCellDB : La base de donn&eacute;es de cellules souches pluripotentes humaines au niveau National Institutes of Health.

Viel von der Aufregung von induzierten pluripotenten Stammzellen-Technologie befasst sich mit der M&ouml;glichkeit der Modellierung sowie das Potenzial f&uuml;r personalisierte Zelltherapie-Krankheit. Um dies zu verfolgen ist es jedoch wichtig zu verstehen, den &#39;normalen&#39; pluripotenten Zustand einschlie&szlig;lich ihrer inh&auml;renten Variabilit&auml;t. Wir haben verschiedene Molekulare Profilerstellungs-Assays f&uuml;r 21 hESC-Linien und 8 HiPSC Zeilen generieren einen umfassenden Snapshot des undifferenzierten pluripotenten Stammzellen durchgef&uuml;hrt. Analyse der Gen-Ausdruck-Daten ergab kein iPSC-spezifische Gen-Ausdruck-Muster nach fr&uuml;heren Berichten. Wir verglichen weitere Zellen, differenzierte als Embryoid K&ouml;rper in 2 Medien vorgeschlagene Differenzierung gegen&uuml;ber separate Zelle Schicksale sowie 20 Erwachsene Gewebe zu initiieren. Aus dieser Analyse haben wir eine gen-Liste erzeugt, die Pluripotenz definiert und etabliert eine Baseline f&uuml;r den pluripotenten Zustand. Schlie&szlig;lich bieten wir Listen von Genen, die unter beiden Differenzierung-Bedingungen, die die vorgeschlagenen in Richtung unabh&auml;ngiger Zelle Schicksale Einseitigkeit bereichert.

StemCellDB: Die menschliche Pluripotent Stem Cell Datenbank an den National Institutes of Health.

Molto l&#39;entusiasmo suscitato dalla tecnologia delle cellule staminali pluripotenti indotte &egrave; interessato la possibilit&agrave; di malattia modellazione cos&igrave; come il potenziale per la terapia cellulare personalizzato. Tuttavia, per perseguire questo &egrave; importante comprendere lo stato &#39;normale&#39; pluripotenti compresa sua variabilit&agrave; intrinseca. Abbiamo eseguito vari saggi di analisi molecolare per 21 linee di hESC e 8 hiPSC per generare uno snapshot completo dello stato indifferenziato di cellule staminali pluripotenti. Analisi dei dati di espressione genica ha rivelato nessun modello di espressione iPSC specifico gene conformemente a precedenti relazioni. Abbiamo confrontato ulteriormente le cellule, differenziate come corpi embryoid in 2 media proposto di avviare la differenziazione verso destini cella separata, come pure i tessuti adulti 20. Da questa analisi abbiamo abbiamo generato un elenco di gene che definisce la pluripotenza e stabilisce una linea di base per lo stato pluripotenti. Infine, forniamo liste di geni arricchiti sotto entrambe le condizioni di differenziazione che mostrano il bias proposto verso destini cellula indipendente.

StemCellDB: L&#39;umano cellule staminali pluripotenti Database presso i National Institutes of Health.

&#x8A98;&#x5C0E;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x6280;&#x8853;&#x306B;&#x3088;&#x3063;&#x3066;&#x751F;&#x6210;&#x3055;&#x308C;&#x305F;&#x8208;&#x596E;&#x306E;&#x591A;&#x304F;&#x306F;&#x30D1;&#x30FC;&#x30BD;&#x30CA;&#x30E9;&#x30A4;&#x30BA;&#x3055;&#x308C;&#x305F;&#x7D30;&#x80DE;&#x7642;&#x6CD5;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3060;&#x3051;&#x3067;&#x306A;&#x304F;&#x3001;&#x30E2;&#x30C7;&#x30EA;&#x30F3;&#x30B0;&#x75BE;&#x60A3;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x3092;&#x61F8;&#x5FF5;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x3053;&#x308C;&#x3092;&#x8FFD;&#x6C42;&#x3059;&#x308B;&#x305D;&#x306E;&#x56FA;&#x6709;&#x306E;&#x5909;&#x52D5;&#x3092;&#x542B;&#x3080; &#39;normal&#39; &#x591A;&#x80FD;&#x6027;&#x306E;&#x72B6;&#x614B;&#x3092;&#x7406;&#x89E3;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x91CD;&#x8981;&#x3067;&#x3059;&#x3002;&#x69D8;&#x3005; &#x306A;&#x5206;&#x5B50;&#x30A2;&#x30C3;&#x30BB;&#x30A4; 21 hESC &#x7DDA;&#x3068;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x672A;&#x5206;&#x5316;&#x72B6;&#x614B;&#x306E;&#x5305;&#x62EC;&#x7684;&#x306A;&#x30B9;&#x30CA;&#x30C3;&#x30D7;&#x30B7;&#x30E7;&#x30C3;&#x30C8;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B; 8 &#x304B;&#x3089;&#x4F5C;&#x88FD;&#x3057;&#x305F;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x884C;&#x3063;&#x305F;&#x3002;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x30C7;&#x30FC;&#x30BF;&#x306E;&#x89E3;&#x6790;&#x306F;&#x3001;&#x4EE5;&#x524D;&#x306E;&#x30EC;&#x30DD;&#x30FC;&#x30C8;&#x306B;&#x5F93;&#x3063;&#x3066; iPSC &#x7279;&#x7570;&#x7684;&#x907A;&#x4F1D;&#x5B50;&#x8868;&#x73FE;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3002;&#x6211;&#x3005; &#x306F;&#x3055;&#x3089;&#x306B;&#x500B;&#x5225;&#x7D30;&#x80DE;&#x904B;&#x547D;&#x3068;&#x3057;&#x3066; 20 &#x6210;&#x4F53;&#x7D44;&#x7E54;&#x3078;&#x306E;&#x5206;&#x5316;&#x3092;&#x958B;&#x59CB;&#x3059;&#x308B;&#x63D0;&#x6848; 2 &#x306E;&#x30E1;&#x30C7;&#x30A3;&#x30A2;&#x306E;&#x80DA;&#x69D8;&#x4F53;&#x3068;&#x3057;&#x3066;&#x5206;&#x5316;&#x3057;&#x305F;&#x7D30;&#x80DE;&#x3092;&#x6BD4;&#x8F03;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x306E;&#x5206;&#x6790;&#x304B;&#x3089;&#x306F;&#x591A;&#x80FD;&#x6027;&#x3092;&#x5B9A;&#x7FA9;&#x3057;&#x3001;&#x591A;&#x80FD;&#x6027;&#x306E;&#x72B6;&#x614B;&#x306E;&#x305F;&#x3081;&#x306E;&#x30D9;&#x30FC;&#x30B9;&#x30E9;&#x30A4;&#x30F3;&#x3092;&#x78BA;&#x7ACB;&#x3059;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x30EA;&#x30B9;&#x30C8;&#x3092;&#x751F;&#x6210;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x63D0;&#x6848;&#x3055;&#x308C;&#x305F;&#x72EC;&#x7ACB;&#x3057;&#x305F;&#x7D30;&#x80DE;&#x904B;&#x547D;&#x30D0;&#x30A4;&#x30A2;&#x30B9;&#x3092;&#x793A;&#x3059;&#x4E21;&#x65B9;&#x306E;&#x5206;&#x5316;&#x306E;&#x6761;&#x4EF6;&#x306E;&#x4E0B;&#x3067;&#x6FC3;&#x7E2E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x30EA;&#x30B9;&#x30C8;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;

StemCellDB&#xFF1A; &#x56FD;&#x7ACB;&#x885B;&#x751F;&#x7814;&#x7A76;&#x6240;&#x3067;&#x306E;&#x3072;&#x3068;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x30C7;&#x30FC;&#x30BF;&#x30D9;&#x30FC;&#x30B9;&#x3002;

&#xC720;&#xB3C4; &#xB9CC;&#xB2A5; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xAE30;&#xC220;&#xC5D0; &#xC758;&#xD574; &#xC0DD;&#xC131; &#xB41C; &#xD765;&#xBD84;&#xC758; &#xC5F4;&#xAE30;&#xAC00; &#xB9CE;&#xC740; &#xC9C8;&#xBCD1; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C;, &#xB9DE;&#xCDA4;&#xD615;&#xB41C; &#xC138;&#xD3EC; &#xCE58;&#xB8CC;&#xC5D0; &#xB300; &#xD55C; &#xAC00;&#xB2A5;&#xC131; &#xBAA8;&#xB378;&#xB9C1;&#xC758; &#xAC00;&#xB2A5;&#xC131;&#xACFC; &#xD568;&#xAED8; &#xC6B0;&#xB824; &#xD558;&#xACE0;&#xC788;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xC774;&#xAC83;&#xC744; &#xCD94;&#xAD6C; &#xD558;&#xB294; &#xADF8;&#xAC83;&#xC740; &#xADF8;&#xAC83;&#xC758; &#xB0B4;&#xC7AC; &#xBCC0;&#xB3D9;&#xC131;&#xC744; &#xD3EC;&#xD568; &#xD55C; &#39;&#xC815;&#xC0C1;&#39; pluripotent &#xC0C1;&#xD0DC;&#xB97C; &#xC774;&#xD574; &#xD558;&#xB294; &#xAC83;&#xC774; &#xC911;&#xC694;&#xC785;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC; 21 hESC &#xB77C;&#xC778;&#xACFC; pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; undifferentiated &#xC0C1;&#xD0DC;&#xC758; &#xC885;&#xD569;&#xC801;&#xC778; &#xC2A4;&#xB0C5;&#xC20F;&#xC744; &#xC0DD;&#xC131; 8 hiPSC &#xB77C;&#xC778;&#xC5D0; &#xB300; &#xD55C; &#xB2E4;&#xC591; &#xD55C; &#xBD84;&#xC790; &#xD504;&#xB85C; &#xD30C;&#xC77C;&#xB9C1; &#xBD84;&#xC11D; &#xC2E4;&#xD5D8;&#xC744; &#xC218;&#xD589; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC720;&#xC804;&#xC790; &#xD45C;&#xD604; &#xB370;&#xC774;&#xD130; &#xBD84;&#xC11D; iPSC &#xD2B9;&#xC815; &#xC720;&#xC804;&#xC790; &#xC2DD; &#xD328;&#xD134;&#xC744; &#xB530;&#xB77C; &#xC774;&#xC804; &#xBCF4;&#xACE0;&#xC11C;&#xB97C; &#xACF5;&#xAC1C; &#xD588;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; &#xB354; &#xC140;, 2 &#xBBF8;&#xB514;&#xC5B4; 20 &#xC131;&#xC778; &#xC870;&#xC9C1;&#xC73C;&#xB85C; &#xBCC4;&#xB3C4; &#xC140; &#xC6B4;&#xBA85;&#xC744; &#xD5A5;&#xD574; &#xCC28;&#xBCC4;&#xD654; &#xC2DC;&#xC791; &#xC81C;&#xC548; embryoid &#xC2DC;&#xCCB4;&#xB85C; &#xCC28;&#xBCC4;&#xD654; &#xB41C; &#xBE44;&#xAD50;. &#xC774; &#xBD84;&#xC11D;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC; &#xC815;&#xC758; pluripotency pluripotent &#xC0C1;&#xD0DC;&#xC5D0; &#xB300; &#xD55C; &#xCD08;&#xAE30; &#xACC4;&#xD68D;&#xC744; &#xC124;&#xC815; &#xD558;&#xB294; &#xC720;&#xC804;&#xC790; &#xBAA9;&#xB85D;&#xC744; &#xC0DD;&#xC131;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C;, &#xC6B0;&#xB9AC;&#xB294; &#xB3C5;&#xB9BD;&#xC801;&#xC778; &#xC138;&#xD3EC; &#xC6B4;&#xBA85; &#xD5A5;&#xD574; &#xC81C;&#xC548; &#xB41C; &#xBC14;&#xC774;&#xC5B4;&#xC2A4;&#xB97C; &#xBCF4;&#xC5EC; &#xB450; &#xBD84;&#xD654; &#xC870;&#xAC74; &#xD558;&#xC5D0;&#xC11C; &#xB18D;&#xCD95; &#xD558;&#xB294; &#xC720;&#xC804;&#xC790;&#xC758; &#xBAA9;&#xB85D;&#xC744; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;.

StemCellDB: &#xC778;&#xAC04;&#xC758; Pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xB370;&#xC774;&#xD130;&#xBCA0;&#xC774;&#xC2A4; &#xAC74;&#xAC15;&#xC758; &#xAD6D;&#xB9BD; &#xC5F0;&#xAD6C;&#xC18C;&#xC5D0;&#xC11C;.

Gran parte de la emoci&oacute;n generada por la tecnolog&iacute;a de c&eacute;lulas madre pluripotentes inducidas se refiere a la posibilidad de enfermedad modelado as&iacute; como el potencial de la terapia celular personalizado. Sin embargo, para dedicarse a esto es importante comprender el estado pluripotente &#39;normal&#39;, incluyendo su variabilidad inherente. Hemos realizado varios ensayos perfiles moleculares para 21 hESCs y 8 hiPSC l&iacute;neas al generar una instant&aacute;nea completa del estado indiferenciado de c&eacute;lulas madre pluripotentes. El an&aacute;lisis de los datos de expresi&oacute;n gen&eacute;tica no revel&oacute; ning&uacute;n patr&oacute;n de expresi&oacute;n de genes espec&iacute;ficos de iPSC seg&uacute;n informes anteriores. Comparamos m&aacute;s c&eacute;lulas diferenciadas como cuerpos embryoid en 2 medios propuestos iniciar la diferenciaci&oacute;n hacia destinos separados de la c&eacute;lula, as&iacute; como 20 tejidos adultos. De este an&aacute;lisis hemos generado una lista del gen que define la pluripotencia y establece un punto de partida para el estado pluripotente. Por &uacute;ltimo, proporcionamos listas de genes enriquecidos en ambas condiciones de diferenciaci&oacute;n que demuestran el sesgo propuesto hacia destinos de c&eacute;lulas independientes.

StemCellDB: El humano Pluripotent Stem Cell base en los institutos nacionales de salud.

Human induced pluripotent stem cells (iPSCs) have great potential as source cells for therapeutic uses. However, reports indicate that iPSCs carry genetic abnormalities, which may impede their medical use. Little is known about mechanisms contributing to intrinsic DNA damage in iPSCs that could lead to genomic instability. In this report, we investigated the level of DNA damage in human iPSC lines compared with their founder fibroblast line and derived mesenchymal stromal cell (MSC) lines using the phosphorylated histone variant, γH2AX, as a marker of DNA damage. We show that human iPSCs have elevated basal levels of γH2AX, which correlate with markers of DNA replication: 5-ethynyl-2'-deoxyuridine and the single-stranded binding protein, replication protein A. γH2AX foci in iPSCs also colocalize to BRCA1 and RAD51, proteins in the homologous repair pathway, implying γH2AX in iPSCs marks sites of double strand breaks. Our study demonstrates an association between increased basal levels of γH2AX and the rapid replication of iPSCs. Stem Cells 2018;36:1501-1513.

High Basal Levels of γH2AX in Human Induced Pluripotent Stem Cells Are Linked to Replication-Associated DNA Damage and Repair.

Use of human pluripotent stem cells (hPSCs) and their differentiated derivatives have led to recent proof-of-principle drug discoveries, defining a pathway to the implementation of hPSC-based drug discovery (hPDD). Current hPDD strategies, however, have inevitable conceptual biases and technological limitations, including the dimensionality of cell-culture methods, cell maturity and functionality, experimental variability, and data reproducibility. In this review, we dissect representative hPDD systems via analysis of hPSC-based 2D-monolayers, 3D culture, and organoids. We discuss mechanisms of drug discovery and drug repurposing, and roles of membrane drug transporters in tissue maturation and hPDD using the example of drugs that target various mutations of CFTR, the cystic fibrosis transmembrane conductance regulator gene, in patients with cystic fibrosis.

Pluripotent Stem Cell Platforms for Drug Discovery.

Anterior-posterior (A-P) specification of the neural tube involves initial acquisition of anterior fate followed by the induction of posterior characteristics in the primitive anterior neuroectoderm. Several morphogens have been implicated in the regulation of A-P neural patterning; however, our understanding of the upstream regulators of these morphogens remains incomplete. Here, we show that the Krüppel-like zinc finger transcription factor GLI-Similar 3 (GLIS3) can direct differentiation of human embryonic stem cells (hESCs) into posterior neural progenitor cells in lieu of the default anterior pathway. Transcriptomic analyses reveal that this switch in cell fate is due to rapid activation of Wingless/Integrated (WNT) signaling pathway. Mechanistically, through genome-wide RNA-Seq, ChIP-Seq, and functional analyses, we show that GLIS3 binds to and directly regulates the transcription of several WNT genes, including the strong posteriorizing factor WNT3A, and that inhibition of WNT signaling is sufficient to abrogate GLIS3-induced posterior specification. Our findings suggest a potential role for GLIS3 in the regulation of A-P specification through direct transcriptional activation of WNT genes. Stem Cells 2018 Stem Cells 2019;37:202-215.

Kyeyoon Park

NIH Stem Cell Unit,
National Institute of Neurological Disorders and Stroke,
NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke

National Institutes of Health

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Kyeyoon Park

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}NIH Stem Cell Unit,National Institute of Neurological Disorders and Stroke,NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke

National Institutes of Health

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

NIH Stem Cell Unit,
National Institute of Neurological Disorders and Stroke,
NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke